Recent work has centered on the study of the mechanism by which diphtheria toxin is secreted from Corynebacterium diphtheriae. We have shown that diphtheria toxin is synthesized exclusively on membrane-bound polysomes and is secreted co-translationally. A restriction endonuclease map of corynephage beta c and the closely related beta vir has been completed using the Eco R-1, Bam H-1, Hind III, and Kpn I enzymes. We have used in vitro 32P-labeled tox messenger RNA to probe restriction digests of beta DNA to physically locate the diphtheria tox operon. The diphtleria tox operon is located on a 3.9 Kb Bam H-1 restriction fragment. The region that specifically hybridizes with the tox mRNA is approximately 2.0 Kb, suggestingg that the diphtheria tox operon is monocistronic.